(1) Field of the Invention
The present invention relates to Novel compounds isolated from the bile of male sea lampreys are described, in particular, 7α,12α,24-trihydroxy-5α-cholan-3-one-24-sulfate and 7α,12α-dihydroxy-5α-cholan-3-one-24-oic acid and an ELISA for detecting the compounds. The bile compounds act as pheromones which attract female sea lampreys in water to the point in the water where the compounds had been introduced. The bile compounds are useful in lamprey population management programs where it is desirable to control the locomotion and distribution of lampreys and in food operations where it enhances the efficiency of catching lampreys for food.
(2) Description of Related Art
The sea lamprey, Petromyzon marinus is an ancestral jawless fish and an invasive parasite of fishes in the Great Lakes of North America. It migrates into streams to spawn in spring. The males arrive earlier than the females (Applegate, U.S. Fish Wild. Serv. Spec. Sci. Rep. Fish. Serv. 55: 237 (1950)) and build nests in areas where flow rates are 0.5 to 1.5 m/s (Applegate, U.S. Fish Wild. Serv. Spec. Sci. Rep. Fish. Serv. 55: 237 (1950)); Manion and Hanson, Can. J. Fish. Aquat. Sci. 37: 1635-1640 (1980)). It has long been suspected that the males release a pheromone to guide the females to their nests (Fontaine, Bull. Sco. Oceanogr. Fr. 17: 1681-1687 (1939); Teeter, Can. J. Fish. Aquat. Sci. 37: 2123-2132 (1980)). This type of sex pheromone, capable of inducing spatial orientation of conspecifics “downwind,” is well-established in insects (Carde and Minks, Annu. Rev. Entomol. 40: 559-585 (1995)), but not so in vertebrates where identified sex pheromones tend to have a small “active space” (See, Novotny et al., Science 231: 722 (1986); Mason et al., Science 245: 290 (1989); Rasmussen et al., Nature 379: 684 (1996); Dulka et al., Nature 325: 251-253 (1987); Sorensen, et al., Biol. Reprod. 39: 1039-1050 (1988)).
In fish, the known sex pheromones are gonadal steroids or prostaglandins and have been identified from a priori knowledge of their structures (Dulka et al., Nature 325: 251 (1987); Sorensen, et al., Biol. Reprod. 39: 1039 (1988); Stacey and Cardwell, in Recent Advances in Marine Biotechnology, Fingemlan, Nagabhushanam, Thompson, eds. (Oxford-IBH Publ., 1997), pp. 407-454).
There is evidence that sex steroids may function as a male sex pheromone in the sea lamprey. Sexually mature females showed strong preference responses to testosterone at concentrations between 3 and 30 pg/L (Adams et al., J. Chem. Ecol. 13: 387-395 (1987)) and sexually mature male sea lampreys are known to release several immunoreactive sex steroids into the water (Linville et al., Horm. Behav. 21: 105-117 (1987); Adams et al., Olfact. Taste 9: 148-151 (1987)). It has long been hypothesized that hormonal pheromones may be common among aquatic animals (Kittredge et al., Fish Bull. (Natl. Mar. Fish. Serv. U.S.) 69: 337-343 (1971)) and all sex pheromones identified to date in teleosts are sex hormones (Stacey and Cardwell, in Recent Advances in Marine Biotechnology, Nagabhushanam and Thompson (eds.), Oxford-IBH, pp. 407-454 (1997)). Testosterone has been suggested to function as a sex pheromone in the Atlantic salmon, Salmo salar (Moore, Proceed. Fourth Intl. Symp. Reprod. Physiol. Fish, Scott et al. (eds.) U. East Anglia Printing Unit, UK, pp. 241-244 (1991)). The biological significance of the observed preference by female sea lampreys for testosterone, however, needs further examination.
The preference response is over a relatively narrow range of concentrations (roughly 10−11 to 10−10 M), which are about 100 times higher than the level of immunoreactive testosterone released by male sea lampreys in behaviorally-active samples (Linville et al., Horm. Behav. 21:105-117 (1987)). Further, immunoreactive testosterone in plasma of sexually mature males is barely measurable (Sower, Fish Physiol. Biochem. 8: 365-374 (1990); Sower et al., Gen. Comp. Endocrinol. 58: 259-269 (1985)). It has been demonstrated that the major androgen in the European river lamprey, Lampetra fluviatilis, is 15β-hydroxytestosterone (Kime and Rafter, In Vitro Gen. Com[. Endocrinol. 44: 69-76 (1981)). If the sea lamprey produces the same androgen, then the differences in immunoreactive testosterone concentrations observed by different workers could be due to differences in the specificity of antibodies against testosterone. This raises the question of whether 15β-hydroxytestosterone, or a metabolite, functions as a pheromone, and if so, is it functionally independent of the bile acid pheromone?
Although sexually mature female lampreys also appear to release a pheromone that attracts male conspecifics (Teeter, Can. J. Fish. Aquat. Sci. 37: 2123-2132 (1980)), the structure of this pheromone has not been identified. The male attracting compound is associated with ovarian fluid of ovulatory females, suggesting that it is synthesized in the ovary and may be related to maturational hormones. The timing of release of the female pheromone is strikingly similar to that of a goldfish female pheromone, prostaglandin F2a (Sorensen et al., Biol. Reprod. 39: 1039-1050 (1988)). However, a direct comparison between lamprey and goldfish is difficult because lampreys are semelparous, having a single spawning period which last for days (Manion and Hanson, Can. J. Fish. Aquat. Sci. 37: 1635-1640 (1980)), whereas, goldfish are iteroparous, with multiple, but short-lived spawning acts, in each spawning season. Consequently, it makes sense that female lamprey pheromone is released over a period of a few days, while female goldfish pheromone is released over a period of hours. Whether biosynthesis of prostaglandins in female sea lampreys can be maintained at a high level over a sustained period in order to function as a pheromone has not been studied. No attempt has been made to fractionate and characterize the structure of the female pheromone in the sea lamprey. It would be interesting to determine whether the female lamprey pheromone is a hormone that is directly associated with ovulation. Maturational hormones have not been studied in the sea lamprey. Plasma levels of immunoreactive progesterone and estradiol are found to increase as the gonads mature (Sower, Fish Physiol. Biochem. 8: 365-374 (1990)). In L. fluviatilis, 15α-hydroxy-progesterone has been identified as the main product when ovaries are incubated with progesterone (Kime and Rafter, In Vitro Gen. Comp. Endocrinol. 44: 69-76 (1981)). Whether this is the maturational hormone for female lampreys has yet to be determined.
Because lampreys are an invasive parasite which have had an adverse effect on particular bodies of water such as the Great lakes basin, there has been a great interest in developing means for controlling lamprey populations. For example, in the Great Lakes basin, sea lampreys are effectively controlled to low levels in most spawning streams using trapping of females and lampricides (Smith and Tibble, Can. J. Fish. Aquat. Sci. 37: 1780-1801 (1980)). There are numerous streams in which sea lampreys continue to spawn, but the density is too low to warrant extensive trapping or lampricide treatment. However, these streams, if left untreated, may become major contributors of parasitic phase lampreys entering the lakes to feed. In addition lampricide compounds are not naturally occurring. Therefore, there is a need for compounds which would optimize the mass trapping of adult females, particularly in areas difficult to treat with lampricide or trap by conventional methods. Preferably, the compounds would be environmentally friendly.